PROJECT SUMMARY During the development of addiction, drugs of abuse induce synaptic plasticity throughout the reward pathways of the brain. Rodent models have been used to show that these changes begin to occur after a single drug administration, enhancing the activity of dopaminergic (DAergic) neurons in the ventral tegmental area (VTA). DA neuron activity is largely regulated by excitatory glutamatergic inputs and inhibitory GABAergic inputs. Recent studies using optogenetic techniques have provided evidence indicating that DAergic cells that project to different targets display different drug-induced glutamatergic plasticity and are differentially involved in the regulation of reward behavior. Though previous studies had suggested that such heterogeneity existed in the VTA, this optogenetic circuit level analysis uniquely provides researchers with the ability to isolate input/output pairs and directly determine their specific synaptic properties. This approach will be used here to investigate morphine effects on synaptic plasticity of local GABAergic inputs onto DAergic neurons projecting to three regions: the lateral nucleus accumbens shell (NAcSh), medial NAcSh, and medial prefrontal cortex (mPFC). Aim 1 - Determine the effect of morphine on VTA GABA synapses onto lateral NAcSh projections Hypothesis: Morphine will decrease the synaptic strength of GABAergic inputs to the lateral NAcSh and block LTPGABA at these synapses. Aim will be addressed by combining AAV2-DIO-YFP-ChR2 injection into the VTA with red fluorescent retrobead injection into the lateral NAcSh of VGAT Cre mice. These animals will be treated with morphine or saline and the plasticity of local GABAergic connections onto retrobead expressing cells will be determined by measuring asynchronous IPSCs (asIPSCs), miniature IPSC (mIPSC) frequency and amplitude, paired pulse ratio (PPR), and LTPGABA. Aim 2 - Determine the effect of morphine of VTA GABA synapses onto medial NAcSh projections Hypothesis: Morphine will either decrease or not change the synaptic strength of GABAergic inputs on medial NAcSh and these synapses will not express LTPGABA. I will perform dual injections of AAV2-DIO-YFP-ChR2 into the VTA and red fluorescent retrobeads into the medial NAcSh of VGAT Cre mice. These animals will be treated with morphine or saline and the plasticity of local GABAergic connections onto retrobead expressing cells will be determined by measuring asIPSCs, mIPSC frequency and amplitude, PPR, and LTPGABA. Aim 3 - Determine the effect of morphine on VTA GABA synapses onto mPFC projections Hypothesis: Morphine will increase the synaptic strength of GABAergic inputs on medial NAcSh but have no effect on LTPGABA. I will perform dual injections of AAV2-DIO-YFP-ChR2 into the VTA and red fluorescent retrobeads into the mPFC of VGAT Cre mice. These animals will be treated with morphine or saline and the plasticity of local GABAergic connections onto retrobead expressing cells will be determined by measuring asIPSCs, mIPSC frequency and amplitude, PPR, and LTPGABA.